Insulin analogs having protracted time action

An insulin analog and analog technology, which can be applied in the direction of insulin, medical preparations containing active ingredients, hormone peptides, etc., can solve problems such as the inability to provide insulin effects.

Inactive Publication Date: 2006-05-31
ELI LILLY & CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The commercial long-acting insulin molecu

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Acylation of A21 with Boc-Arg(Pbf)-NHS Ester Gly B31 Arg B32 Arg - Human insulin production

[0105] A0 Arg A21 Gly B31 Arg B32 Arg - human insulin

[0106] Boc-Arg(Pbf)-OH, N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC) each 0.4mml mixed in dichloromethane for 30 minutes to prepare Boc-Arg(Pbf)-NHS Esters (0.4 mmol). After filtration, the reaction mixture was concentrated to dryness in a rotary evaporator and dissolved in 4 ml MeOH. 180mg A21 Gly B31 Arg B32 Arg - Human insulin (0.030mmol) dissolved in 20ml containing 50mmol Na 2 HPO 4 50:50 water / CH 3In CN, adjust the pH to 7.7 with 5M KOH solution. To the insulin solution was added 1.2 ml of Boc-Arg(Pbf)-NHS ester solution (0.12 mmol). The reaction was stirred at room temperature for 50 minutes, then an additional 1.2 ml of Boc-Arg(Pbf)-NHS ester solution (0.12 mmol) was added and allowed to react for an additional 30 minutes, followed by acidification with 100 μl of TFA. Sa...

Embodiment 2

[0109] In vitro receptor affinity

[0110] The affinity of insulin analogues for the human insulin receptor (IR) was determined using a radiolabeled ligand [ 125 I] Determined in a competitive binding assay for insulin. Human insulin receptor membranes were prepared from P1 membranes of stably transfected 293EBNA cells overexpressing the receptor. The analytical method was carried out and validated in filtration and SPA (Scintillation Proximity Assay) modes which gave comparable results, but with WGA PVT PEI SPA beads treated with PVT PEI from AmershamPharmacia Biotech., type A (WGA PVT PEI SPA) beads in the SPA mode.

[0111] Radiolabeled ligand ([ 125 I] Recombinant human insulin) can be self-made or purchased from AmershamPharmacia Biotech. Company, the specific radioactivity of the reference date is 2000Ci / mmol. The SPA assay buffer was 50 mM Tris-HCL, pH 7.8, 150 mM NaCl, 0.1% BSA. Assays were automated in a high-throughput setup in 96-well micropl...

Embodiment 3

[0122] In vitro metabolic capacity

[0123] A0 Arg A21 Gly B31 Arg B32 Arg - The metabolic capacity (glucose uptake) of human insulin and recombinant human insulin was determined in a glucose uptake assay using differentiated mouse 3T3-L1 adipocytes. At a density of 25,000 cells / well in 100 μl growth medium (DMEM high glucose without L-glutamine, 10% calf serum, 2 mM L-glutamine, 1% antibiotic / antifungal solution) Undifferentiated mouse 3T3 cells were plated.

[0124] 3 days after plating by adding differentiation medium: high glucose and L-glutamine-free DMEM, 10% FBS, 2mM L-glutamine, 1% antibiotic / antifungal solution, 10mM HEPES, 0.25mM dexamethasone, Differentiation was initiated with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 5 mg / ml insulin. After 48 hours (day 3), the original differentiation medium was changed to a differentiation medium containing insulin but without IBMX or dexamethasone and by day 6 the cells were switched to a differentia...

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PUM

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Abstract

The present invention provides insulin analogues A0ArgA21GlyB31ArgB32Arg that provide a prolonged and smooth basal duration of action. The invention also provides a method of treating diabetes comprising administering an insulin analog.

Description

[0001] This application claims priority to U.S. Provisional Application No. 60 / 466,501, filed April 29, 2003, U.S. Provisional Application No. 60 / 466,500, filed April 29, 2003, U.S. Provisional Application No. 60 / 466,500, filed May 13, 2003 Priority of Provisional Application No. 60 / 470,118, each of which is hereby incorporated by reference. field of invention [0002] The present invention relates to insulin analogues useful in the treatment of hyperglycemia characteristic of diabetes. Background of the invention [0003] The physiological need for insulin can be divided into two phases: a) the nutrient absorption phase, which requires pulses of insulin to break down the meal-related blood glucose surge, and b) the post-absorption phase, which requires a sustained release of insulin to regulate Hepatic glucose output to maintain optimal fasting blood glucose levels, also known as "basal" insulin secretion. [0004] Effective insulin therapy for people with diabetes usually...

Claims

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Application Information

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IPC IPC(8): C07K14/62A61K38/28
Inventor R·D·迪马其W·D·科恩张连山
Owner ELI LILLY & CO
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